Protein therapeutics are an important part of drug discovery. High-throughput screening of large libraries of polynucleotides encoding protein variants, which may include antigen binding polypeptides or fragments thereof, allows efficient discovery or optimization of protein therapeutics for desirable properties such as binding affinity, avidity, stability and specificity. Typical tools used for high-throughput screen include phage and ribosome display techniques.
Therapeutic antigen binding polypeptides or fragments are particularly attractive because of their high affinity and specificity to the antigen and because of their relatively high stability in vitro and in vivo. Antibodies are made of two heavy and two light chains, which contain the variable regions at their N-terminus and are linked by disulfide bridges. Single-chain antibodies in particular have been engineered by linking fragments of the variable heavy and light chain regions (ScFv) into a single polypeptide.
Typical procedures for making ScFv generally involve amplification of gene regions that encode the variable regions of the antibodies, assembly of an ScFv genetic sequence and expression of the ScFv polypeptide sequence in host cells. The host cells are screened using a target polypeptide of interest to identify those cells that express a ScFv polypeptide that binds to this target polypeptide. The host cells can subsequently be analyzed for the polynucleotide coding sequence encoding the expressed ScFv. The most commonly used techniques to identify single-chain antibodies that bind specific polypeptides is by phage display and variations thereof (see Hoogenboom et al., 1998). Generally, phage display methods involve the insertion of random heterologous polynucleotides into a phage genome such that they direct a bacterial host to express peptide libraries fused to phage coat proteins (e.g., filamentous phage pIII, pVI or pVIII). Libraries of up to 1010 individual members can be routinely prepared and screened in this way. Incorporation of the ScFv sequences into the mature phage coat sequence results in the ScFv antibodies encoded by the heterologous polynucleotide sequence being displayed on the exterior surface of the phage. By immobilizing a relevant polypeptide target (or targets) of interest to a surface, a phage that expresses and displays an ScFv that binds to one of those targets on the surface will remain bound while others are removed by washing.